Block copolypeptide, polymer fiber, and preparation method therefor and use thereof

Abstract

Disclosed in the present application are a block copolypeptide, a polymer fiber, and a preparation method therefor and the use thereof. The block copolypeptide comprises a dendritic polymer and a polypeptide chain linked to the dendritic polymer, wherein the polypeptide chain consists of a structural unit as represented by formula (I) and a structural unit as represented by formula (II), the dendritic polymer is linked to the structural unit as represented by formula (I) via an amide bond, and the structural unit as represented by formula (I) is linked to the structural unit as represented by formula (II) via an amide bond. The block copolypeptide of the present application enables functional units which would otherwise form a β-sheet conformation to undergo chain propagation in a metastable α-helix state, and subsequently converts the metastable α-helix into a β-sheet, thus exhibiting a good stability. The formed polymer fiber has superior mechanical properties, and can be widely used in key fields such as high-toughness composite materials, degradable biomedical materials, and intelligent drug delivery systems.

Description

A block copolymerized polypeptide, polymer fiber and preparation method and use thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of polymer synthesis, in particular to a block copolymerized polypeptide, polymer fiber and preparation method and use thereof. BACKGROUND

[0002] Spider silk-like proteins have high strength, high toughness and excellent biocompatibility, and can be widely used in the design and research and development of high-toughness high-strength materials, the development of high-end biomedical engineering instruments, the construction of high-efficiency drug delivery systems, and other fields, showing great commercial application prospects. Spider silk-like proteins are a kind of artificial synthetic high-molecular-weight polypeptide material, whose structure and properties imitate natural spider silk proteins. Researchers have found that the crystalline region of natural spider silk is the key to the tensile strength of spider silk fibers, especially the beta-sheet structure. At present, it is usually prepared by genetic engineering recombinant expression, chemical synthesis or other molecular engineering techniques.

[0003] The genetic engineering recombinant expression method for preparing spider silk-like proteins is one of the most popular and concerned technical routes. The core of this method is to transfer the plasmid into a microbial host to achieve efficient expression of the target protein. However, the natural spider silk protein gene usually has the following characteristics: (1) contains a highly repetitive long sequence; (2) contains non-standard codon bias characteristics, which makes it challenging to efficiently transcribe and translate in a heterologous expression system (such as bacteria, yeast or insect cells). In addition, since the spider silk protein gene is usually large (often more than 10 kb), it also poses a technical limitation to traditional cloning methods. In terms of structural characteristics, the ordered secondary structure of spider silk protein (such as beta-sheet or alpha-helix) needs specific conditions to achieve the correct three-dimensional conformation. Unfortunately, widely used expression hosts such as E. coli lack sufficient chaperone systems and post-translational modification mechanisms, making it difficult to ensure that the target protein obtains the ideal spatial structure. This deficiency often leads to protein aggregation, severely restricting the preparation of soluble and functional spider silk-like proteins. Chemical synthesis is another commonly used method to obtain specific sequence polypeptides. Solid-phase synthesis technology can provide precise control of amino acid sequences, but its low efficiency limits the obtainment of high-molecular-weight spider silk-like proteins. Although N-carboxylic anhydride (NCA) ring-opening polymerization and chemical enzyme-catalyzed polymerization provide new ways to prepare the desired structure, when synthesizing stereospecific beta-sheet polypeptides, these systems often easily produce precipitates, leading to reaction inactivation, making it difficult to obtain high-molecular-weight, high-beta-sheet content spider silk-like proteins. The content of beta-sheet in natural spider silk is 18.3%.

[0004] In addition, the spider silk-like proteins are formed into spider silk-like fibers through a spinning process. In the spinning process, the self-assembly behavior of the spider silk-like proteins is affected by various environmental factors (such as pH, ionic strength, and temperature). How to simulate the special conditions (high concentration, low ion environment) in the spider gland to achieve efficient protein self-assembly and functional regulation is still a technical problem that has not been completely solved.

[0005] The most widely used method for ring-opening polymerization of N-carboxyanhydride (NCA) is to use primary amines, n-hexylamine, hexamethyl disilazane (HMDS), bispyridyl nickel (COD) as an initiator, and DMF as a solvent to open the ring of NCA. NCA is a cyclic amino acid derivative, and its general structure is as follows: The instability of NCA monomers to water and the side reactions accompanying long-term polymerization limit the production of high molecular weight, narrow distribution poly-peptide materials. For example, CN118240229A discloses a dendritic polyamino acid, a preparation method thereof, a nano adjuvant, and a nano vaccine. The dendritic polymer or branched polymer unit is connected with a polyamino acid unit. However, the applicant found in later research that if the dendritic polymer or branched polymer unit is connected with amino acids (glutamic acid, lysine, arginine, glutamine, methionine, and their side chain modified derivatives) that are prone to form alpha-helices, dendritic polyamino acids can be obtained, but fibers cannot be formed using them later. If the dendritic polymer or branched polymer unit is connected with amino acids (such as alanine, serine, tyrosine, phenylalanine, valine, threonine, tryptophan, isoleucine, and their side chain modified derivatives) that are prone to form beta-pleated sheets, the polyamino acid unit is prone to form beta-pleated secondary structures through hydrogen bonds during the synthesis process, resulting in intermolecular aggregation and precipitation. The end amino groups of the polyamino acid unit are inactivated due to the precipitation of the polymerization product, and more amino acid units cannot be connected. The degree of polymerization is less than 10.

[0006] Therefore, it is of great research significance and application value to develop a method that can obtain a block copolymer polypeptide with high solid content, high molecular weight, and high beta-pleated sheet content, further form a polymer fiber, and effectively regulate the fiber in an in vitro environment and realize the construction of a multi-level structure. SUMMARY

[0007] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a block copolymer polypeptide, a polymer fiber, and a preparation method and use thereof.

[0008] To achieve the above-mentioned objects and other related objects, the present application is obtained by the following technical solutions.

[0009] A first aspect of the present invention protects a block copolymer polypeptide comprising a dendritic polymer and a polypeptide segment connected to the dendritic polymer, the polypeptide segment being composed of structural units shown in Formula I and structural units shown in Formula II, wherein the dendritic polymer is connected to the structural unit shown in Formula I via amide bonds, and the structural unit shown in Formula I is connected to the structural unit shown in Formula II via amide bonds.

[0010] In Formula I, R1 is selected from the side groups of the following amino acids: glutamic acid, lysine, arginine, glutamine, and methionine or their derivatives;

[0011] In Formula I, R2 is selected from the side groups of the following amino acids: alanine, serine, tyrosine, phenylalanine, valine, threonine, tryptophan, and isoleucine or their derivatives.

[0012] In this application, R1 contains a functional unit that readily forms an α-helix / random coil conformation, and R2 contains a functional unit that readily forms a β-sheet conformation. The block copolymer polypeptide of this application has a dendritic polymer core, with its terminal amino groups linked to polypeptide segments. These polypeptide segments are polymerized from amino acids containing functional units readily forming α-helical / random coil conformations and amino acids containing functional units readily forming β-sheet conformations. The introduction of functional units with α-helical / random coil conformations allows the polypeptide chain, which would otherwise form a β-sheet conformation, to grow in a metastable α-helix form, and the conformation is tunable.

[0013] During post-processing, conformational changes can occur. For example, by using specific solvents such as DMF (dimethylformamide) or methanol, infrared spectroscopy reveals a significant increase in absorbance of the β-sheet region after the addition of DMF or methanol, indicating that the metastable α-helix conformation is induced to change and form a β-sheet. Conformational changes can also be induced by mechanical stretching. For instance, in polymer fibers formed from block copolymers, the peak intensity corresponding to the β-sheet significantly increases with the increase of the stretching ratio during stretching, and the content increases from 30% to 60%, while the characteristic peak of the α-helix relatively weakens and the content decreases from 80% to 20%, indicating that the metastable α-helix conformation of the block copolymer is induced to change and form a β-sheet.

[0014] The structural design of the block copolymer of this invention significantly increases the β-sheet content, providing a new pathway for forming polymer fibers (artificial spider silk). Furthermore, the solvent-induced conformational change mechanism not only provides flexibility for the processing and application of the block copolymer but also expands its functional properties under different environments. Simultaneously, the block copolymer of this application exhibits good property stability; its viscosity and modulus did not change significantly within 15 days, and its metastable α-helices did not assemble or transform into β-sheets. In summary, the block copolymer of this application, with its unique structural design, tunable conformational change, and stable properties, shows broad application prospects in biomedicine, materials science, and other fields.

[0015] In some embodiments, the block copolymer polypeptide has the following general formula:

[0016] Where m is independently 10 to 1000, and n is independently 10 to 1000;

[0017] R is a dendritic polymer.

[0018] In some implementations, m can be 10-1000, 10-400, 200-600, 700-1000, or 10, 50, 100, 400, 600, or 1000.

[0019] In some implementations, n can be 10 to 1000, 10 to 400, 200 to 600, 700 to 1000, or 10, 50, 100, 400, 600, or 1000.

[0020] In some implementations, m and n can be the same or different. m and n can be obtained by adjusting the molar ratio of NCA in the reaction.

[0021] In some embodiments, the dendritic polymer is selected from one or more of polyamide-amine (PAMAM), polypropyleneimide (PPI), and branched polyethyleneimine (PEI). Dendritic polymers are macromolecules with a dendritic structure, formed by the repeating and linear linkage of oligomers through branching units. They typically consist of a core, a polymer backbone, and side chains of branching units, and are monodisperse polymers with a highly branched structure. They can provide a large number of amino-terminal active sites, thereby improving the conversion efficiency of amino acid N-carboxyl anhydride monomers. Preferably, it is polyamide-amine (PAMAM). The polyamide-amine (PAMAM) is of generation 0 to 9, such as first-generation PAMAM (G1-PAMAM), second-generation PAMAM (G2-PAMAM), third-generation PAMAM (G3-PAMAM), fourth-generation PAMAM (G4-PAMAM), fifth-generation PAMAM (G5-PAMAM), sixth-generation PAMAM (G6-PAMAM), seventh-generation PAMAM (G7-PAMAM), eighth-generation PAMAM (G8-PAMAM), and ninth-generation PAMAM (G9-PAMAM). The applicant has studied polyamide-amines of different generations, and all of them can be used to prepare block copolymer peptides.

[0022] In some embodiments, the weight-average molecular weight of the block copolymer is 1-10000 kDa, or 1-3000 kDa, or 2000-6000 kDa, or 5000-10000 kDa, or 2349, 2541, 2042, or 3028 kDa.

[0023] In some embodiments, the derivative is a derivative derived from an amino acid; the derivative includes the introduction or substitution of functional groups in the main chain or side chain of the amino acid. The functional groups include, but are not limited to, hydroxyl, alkyl, amino, carboxyl, and protecting groups. The alkyl groups include, but are not limited to, methyl and ethyl groups. The protecting groups include, but are not limited to, benzoyl (Cbz), tert-butoxycarbonyl (Boc), and 9-fluorenylmethoxycarbonyl (Fmoc). Common derivatives of amino acid NCA include: O-benzyl ester-L-glutamic acid-N-carboxylic anhydride (BLG-NCA), O-tert-butoxycarbonyl-L-glutamic acid-N-carboxylic anhydride (tBu-BLG-NCA), O-benzyl ester-L-tyrosine-N-carboxylic anhydride (BLT-NCA), O-tert-butoxycarbonyl-L-tyrosine-N-carboxylic anhydride (tBu-BLT-NCA), O-benzyl ester-L-serine-N-carboxylic anhydride (BLS-NCA), N6-benzyloxycarbonyl-L-lysine intracyclic anhydride, etc.

[0024] Another aspect of the present invention protects a method for preparing the block copolymer polypeptide as described above, comprising the following steps:

[0025] 1) The dendritic polymer and the first amino acid N-carboxylic acid anhydride undergo a first ring-opening polymerization reaction in the first solvent to obtain the polymer product;

[0026] 2) The polymerization product and the second amino acid N-carboxylic anhydride undergo a second ring-opening polymerization reaction in a second solvent to obtain the block copolymer polypeptide;

[0027] The amino acid in the first amino acid N-carboxylate anhydride is selected from glutamic acid, lysine, arginine, glutamine, and methionine or their derivatives; the amino acid in the second amino acid N-carboxylate anhydride is selected from alanine, serine, tyrosine, phenylalanine, valine, threonine, tryptophan, and isoleucine or their derivatives.

[0028] This application uses dendritic polymers as initiators. On the one hand, the branched structure of dendritic polymers can provide a large number of amino-terminal active sites, thereby improving the conversion efficiency of the first amino acid N-carboxylate monomer. Compared with using linear n-hexylamine as an initiator and without adding a catalyst in the solvent system, the time of the first ring-opening polymerization reaction in this application is shortened from 2-6 h to 10 min under the same conditions. On the other hand, through the first ring-opening polymerization reaction, the first amino acid N-carboxylate (first NCA) containing functional units that easily form α-helical or uncoiled conformations is introduced to the amino terminus of the dendritic polymer. The resulting polymer product is also dendritic with active amino groups at the end, which can continue to initiate the second ring-opening polymerization of the second amino acid N-carboxylate (second NCA) containing units that easily form β-sheet conformations, allowing the polypeptide chain to grow rapidly in a metastable α-helical form. This application solves the technical problem in the prior art that when dendritic polymers react with N-carboxylate anhydrides (second NCA) containing a second amino acid that easily forms a β-sheet conformation, precipitation is easily formed due to the large number of hydrogen bonds or hydrophobic side chains between the molecular chains of the second amino acid N-carboxylate anhydride (second NCA). Furthermore, the precipitation causes the terminal amino groups of the second NCA to become deactivated, thus preventing the connection of more NCA monomers.

[0029] In some embodiments, the polymer product (dendritic helical macromolecule) obtained from the first ring-opening polymerization reaction not only protects the N-carboxylate anhydride of the second amino acid, preventing direct reaction between the N-carboxylate anhydride and the initiator to produce precipitation, but also catalyzes the ring-opening polymerization of the N-carboxylate anhydride. That is, it maintains the activity of the terminal amino group of the N-carboxylate anhydride, achieving efficient ring-opening polymerization and introducing a convertible β-sheet block. Furthermore, this application uses an amino acid containing an amino acid readily forming an α-helical conformation for the first ring-opening polymerization and an amino acid containing an amino acid readily forming a β-sheet conformation for the second polymerization, allowing the N-carboxylate anhydride of the second amino acid to achieve rapid chain growth through the α-helical conformation of the N-carboxylate anhydride of the first amino acid. Further, the β-sheet block of this application maintains a metastable α-helical structure during synthesis, but transforms in situ into β-sheet nanocrystals during solvent displacement and stretching. Furthermore, this application utilizes the characteristic of the first amino acid N-carboxylic acid anhydride to catalyze its own polymerization to participate in controlling the conformational evolution of the polypeptide chain.

[0030] In some embodiments, the dendritic polymer is selected from one or more of polyamide-amine, polypropyleneimide, and branched polyethyleneimine.

[0031] In some embodiments, the amino acid in the first amino acid N-carboxylate is selected from one or more of glutamic acid, lysine, arginine, glutamine, and methionine or their derivatives. The amino acid in the first amino acid N-carboxylate contains a functional unit that readily forms an α-helix or random coil conformation. The amino acid can be derived from a natural amino acid sequence or a non-natural amino acid sequence synthesized artificially.

[0032] In some embodiments, the amino acid in the second amino acid N-carboxylate anhydride is selected from one or more of alanine, serine, tyrosine, phenylalanine, valine, threonine, tryptophan, and isoleucine or their derivatives. The amino acid in the second amino acid N-carboxylate anhydride is an amino acid containing a functional unit that readily forms a β-sheet conformation.

[0033] In some embodiments, the first solvent is selected from one or both of dichloromethane and chloroform.

[0034] In some embodiments, the first solvent is selected from dichloromethane.

[0035] In some embodiments, the second solvent is selected from one or both of dichloromethane and chloroform.

[0036] In some embodiments, the second solvent is selected from dichloromethane.

[0037] In some embodiments, the molar mass ratio of the dendritic polymer to the first amino acid N-carboxylic anhydride is 1:(15-400), and can also be 1:(15-80), 1:(50-150), 1:(110-220), 1:(210-350), 1:(280-400), or 1:50 or 1:10.

[0038] In some embodiments, the molar mass ratio of the dendritic polymer to the second amino acid N-carboxylic anhydride is 1:(15-400), and can also be 1:(15-80), 1:(50-150), 1:(110-220), 1:(210-350), 1:(280-400), or 1:50 or 1:10.

[0039] In some embodiments, the temperature of the first ring-opening polymerization reaction is 10°C to 65°C, or it can be 10°C to 20°C, 20°C to 30°C, 30°C to 40°C, 40°C to 50°C, 50°C to 65°C, or it can be 25°C.

[0040] In some embodiments, the temperature of the second ring-opening polymerization reaction is 10°C to 65°C, or it can be 10°C to 20°C, 20°C to 30°C, 30°C to 40°C, 40°C to 50°C, 50°C to 65°C, or it can be 25°C.

[0041] In some embodiments, the method includes sequentially adding a first amino acid N-carboxylate and a second amino acid N-carboxylate to perform multiple ring-opening polymerization reactions. After multiple ring-opening polymerization reactions, a multi-block copolymer polypeptide with alternating α-helical and β-sheet secondary structures is obtained.

[0042] In some embodiments, the method yields a block copolymer polypeptide solution with a solid content of 15–70 wt%. This invention can obtain high-solid-content block copolymer polypeptides by changing the NCA monomer concentration (0.05 M–2.5 M) and the number or molecular weight of polymerized blocks, which can be directly used for subsequent spinning without requiring a series of operations such as precipitation, purification, reconstitution, and concentration.

[0043] Another aspect of the present invention protects a polymer fiber, the raw material of which comprises the block copolymer polypeptide as described above. The polymer fiber of this application contains a high content of β-sheets.

[0044] In some embodiments, the β-sheet content in the polymer fiber is 1-65%, or it can be 1-28%, or it can be 25-45%, or it can be 40-65%. In this application, the β-sheet content is collected by Fourier transform infrared spectroscopy (scanning range 4000-400cm). -1 The scanning resolution is 4cm. -1 (The number of scans was 16). Then, the peak deconvolution integral of the amide I region of the polymer fiber was performed using the second derivative method, and the content of β-sheet in the polymer fiber was estimated by fitting a Gaussian model. The reference for the Gaussian model fitting can be found in J.Am.Chem.Soc.2024,146,46,31849-31859.

[0045] In some embodiments, the tensile strength of the polymer fiber is 30-220 MPa, or it can be 30-120 MPa, or it can be 100-160 MPa, or it can be 140-220 MPa, or it can be 50, 78, 80, 100, 156, 175, or 200 MPa.

[0046] In some embodiments, the strain of the polymer fiber is 30-150%, or it can be 30-150%, or it can be 30-150%, or it can be 30, 40, 48, or 140%.

[0047] The single polymer fiber of this application exhibits a regular cylindrical morphology and is essentially a bundle structure composed of hundreds of microscopic nanofibers with diameters ranging from 50 to 200 nanometers, arranged parallel along the axial direction, forming a unique multi-layered structure. Crystallographic observations reveal the presence of large, parallel-aligned β-folded lamellar crystals within the fiber. This fine internal structure indicates that the prepared polymer fiber can mimic the formation mechanism of artificial spider silk and can be used to construct multi-layered artificial spider silk.

[0048] Another aspect of the present invention protects the method for preparing polymer fibers as described above, wherein the polymer fibers are obtained by spinning a block copolymer polypeptide solution.

[0049] In some embodiments, the process includes the following steps: mixing a block copolymer peptide solution into a coagulation bath for treatment, followed by stretching and drying to obtain the polymer fiber. This application employs coagulation bath-induced phase separation technology and stretching-induced orientation crystallization technology to achieve the formation of a multi-level fiber structure from the block copolymer peptide via a micellar nucleation-assembly pathway. Ultimately, a polymer fiber with a multi-level ordered structure was successfully prepared, overcoming limitations such as traditional preparation processes and difficulties in structural control. The multi-level ordered structure construction of polymer fibers (artificial spider silk material) was successfully achieved, specifically from the self-assembly of molecular-level amino acid chains (tyrosine modules) into rigid β-sheet crystals (providing strength) and flexible random coils (contributing toughness), to the parallel binding of nanofibers, ultimately forming a macroscopic polymer fiber material. This hierarchical design is like the synergy of a "molecular spring and a steel plate": the β-crystal resists stretching, and the amorphous region absorbs impact energy through chain segment defolding, thereby improving mechanical strength.

[0050] In some embodiments, the block copolymer peptide solution is mixed into the coagulation bath by a shearing method. The shearing method in this application includes, but is not limited to, using a syringe pump to push the spinning solution at a constant speed. The extrusion rate of the block copolymer peptide solution is 5–50 mL / min. This invention uses a shear flow field to initiate the assembly of the block copolymer peptide into amorphous precursor fibers in the spinning coagulation bath, and in the subsequent drawing process, the convertible β-sheet block (metastable α-helical conformation during synthesis) obtained by the ring-opening polymerization of the second amino acid N-carboxylic anhydride is transformed into β-sheet nanocrystals through in-situ polymerization.

[0051] In some embodiments, the solid content of the block copolymeric peptide solution is 15–70 wt%. Preferably, it is 60–70 wt%.

[0052] In some embodiments, the coagulation bath is selected from one or more of methanol, N,N-dimethylformamide (DMF), ethanol, tetrahydrofuran, ethyl acetate, dimethyl sulfoxide, dichloromethane, and hexafluoroisopropanol.

[0053] In some specific embodiments, the coagulation bath is a mixture of ethanol and tetrahydrofuran, or a mixture of ethyl acetate and dimethyl sulfoxide, or a mixture of methanol and N,N-dimethylformamide. The proportion of solvents in the mixture can be adjusted by those skilled in the art according to actual needs. Preferably, the volume ratio of methanol to N,N-dimethylformamide can be (1–6):1, (1–3):1, (2.5–5.2):1, (3.5–6):1, or 3:1.

[0054] In some embodiments, the processing temperature is 10°C to 35°C, and may also be 10°C to 18°C, 15°C to 26°C, 22°C to 35°C, or 25°C.

[0055] In some embodiments, the stretching ratio is 0.5 to 5 times. Preferably, it is 1 to 3 times.

[0056] In some embodiments, the stretching rate is 1 to 10 mm / s. Preferably, it is 2 mm / s.

[0057] In some embodiments, the drying temperature is 10–160°C. Preferably, it is 20–40°C.

[0058] Another aspect of the present invention protects the use of the block copolymer polypeptides or polymer fibers as described above in fiber products, biomedical materials or drug delivery.

[0059] The block copolymer polypeptides or polymer fibers of this application, due to their unique structural design, can connect more amino acids containing readily β-sheets to form a metastable α-helix conformation. Through solvent extraction or stretching, the metastable α-helix conformation can be transformed into a β-sheet conformation. They also possess excellent mechanical properties and good biocompatibility, making them widely applicable in the design and development of high-toughness and high-strength materials, the development of high-end biomedical engineering devices, and the construction of efficient drug delivery systems, demonstrating enormous commercial application potential. Compared with existing technologies, this invention has the following beneficial effects:

[0060] 1) The block copolymer of this application uses a dendritic polymer as its core, with terminal amino groups linked to polypeptide segments. These segments are polymerized from amino acids containing functional units that readily form α-helical / random coil conformations and amino acids containing functional units that readily form β-sheet conformations. The introduction of functional units readily forming α-helical / random coil conformations into the first segment of the NCA linked by the dendritic polymer causes the polypeptide chain, which would otherwise form a β-sheet conformation, to grow in a metastable α-helix form, and the conformation is tunable. Later, solvent induction or stretching can transform the metastable α-helix conformation into a β-sheet, forming oriented β-sheet nanocrystals. This increases the β-sheet content in the block copolymer. Furthermore, the block copolymer of this application does not self-assemble into a gel or transform into a β-sheet after 15 days at room temperature, indicating extremely high stability.

[0061] 2) By controlling the sequence of ring-opening polymerization of amino acid N-carboxylic anhydride and dendritic polymer, this invention has successfully developed a spider silk-like material with high solid content, high molecular weight, and high β-sheet content.

[0062] 3) This application employs coagulation bath solvent-induced phase separation technology and stretching-induced orientation crystallization technology to achieve effective control over the nucleation and self-assembly of simplified spider silk-like multi-block aggregate micelles with alternating α-helical and β-fold secondary structures at both the micro and macro scales, thereby successfully preparing polymer fibers with multi-level ordered structures (structures similar to artificial spider silk materials).

[0063] 4) The block copolymer polypeptides and polymer fibers of the present invention have high β-sheet content and high mechanical properties, which will strongly promote technological breakthroughs in key fields such as high-toughness composite materials, biodegradable biomedical materials and intelligent drug delivery systems. Attached Figure Description

[0064] Figure 1 shows G3-PAMAM-PBLG 50 -b-PVAL 50 and G3-PAMAM-PVAL 50 Infrared structural diagram.

[0065] Figure 2 shows G3-(PBLG) 50 -b-PBLT 50 )4 Synthesis route diagram.

[0066] Figure 3 shows G3-(PBLG) with different numbers of blocks. 50 -b-PBLT 50 ) n The gel chromatogram is also known as the molecular weight chromatogram.

[0067] Figure 4 shows the high solids content G3-(PBLG) 50 -b-PBLT 50 )4. Stability test of spinning solution, i.e., rheological strain scanning diagram.

[0068] Figure 5 shows G3-PBLG 10 and G3-PBLG 10 -b-PBLT 10 -b-PBLG 10 The gel chromatogram is also known as the molecular weight chromatogram.

[0069] Figure 6 shows G3-PAMAM-(PBLG) with low solids content. 50 -b-PBLT 50 Infrared spectra of dichloromethane reaction solution before and after DMF treatment for 2 hours.

[0070] Figure 7 shows G3-PAMAM-(PBLG) 50 -b-PBLT 50 4. Photographs of fiber samples before and after stretching (top: 0X, bottom: 2X).

[0071] Figure 8 shows G3-PAMAM-(PBLG) 50 -b-PBLT 50 4. Scanning electron microscope images of the cross-section and longitudinal section of the fiber sample.

[0072] Figure 9 shows G3-PAMAM-(PBLG) 50 -b-PBLT 50 )4 Polarizing microscope test image of fiber sample.

[0073] Figure 10 shows G3-PAMAM-(PBLG) 50 -b-PBLT 50 4. Infrared spectra of fiber samples at different draw ratios and their peak fitting analysis.

[0074] Figure 11 shows G3-PAMAM-(PBLG) 50 -b-PBLT 50 )4. Mechanical property data of fiber samples at different draw ratios.

[0075] Figure 12 shows the mechanical properties of fiber samples with different double-ended secondary structures and block sequence fibers at a stretching ratio of 2.

[0076] Figure 13 shows G3-PAMAM-PBLG 400 Photograph of the fiber sample after spinning.

[0077] Figure 14 shows G3-PAMAM-PBLG 400 A real photo of the solution being squeezed into the coagulation bath. Detailed Implementation

[0078] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0079] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0080] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0081] The device information in the following embodiments of this application is as follows:

[0082] Fourier transform infrared spectroscopy (FTIR, Bruker, Karlsruhe, Germany), atomic force microscopy (AFM, Oxford Instruments, Jupiter XR), tensile strength tester (CellScale, UniVert, Canada), Leica sputtering system (Leica EM ACE600, Germany), field emission scanning electron microscope (Gemini 450, Germany, Zeiss).

[0083] O-Benzyl-L-glutamic acid-N-carboxylic acid anhydride (BLG-NCA), containing functional units that readily form α-helix / random coil conformations, has CAS number 3190-71-4 and its structure is as follows:

[0084] L-valine-N-carboxylic acid anhydride (VAL-NCA), containing a functional unit that readily forms a β-sheet conformation, has CAS number 24601-74-9 and its structure is as follows:

[0085] O-Benzyl-L-tyrosine-N-carboxylic acid anhydride (BLT-NCA), containing a functional unit that readily forms a β-sheet conformation, has the CAS number 2439-82-9 and its structure is as follows:

[0086] In the following examples, G3-PAMAM (i.e., a third-generation polyamide-amine dendritic polymer) was used as an example for the preparation of block copolymer peptides, but it is not limited to G3-PAMAM. G3-PAMAM is a transparent liquid with good flowability. It was purchased from the Shanghai branch of Beijing Innocare Technology Co., Ltd., CAS number 153891-46-4, with a weight-average molecular weight of 6848.79. Its structural formula is as follows:

[0087] In the following embodiments of this application, the ring-opening polymerization reaction was carried out at room temperature, which was 25°C.

[0088] Example 1

[0089] This embodiment 1 provides a block copolymer and a method for preparing the block copolymer, including the following steps:

[0090] 1) First ring-opening polymerization: 1.25 μL of G3-PAMAM was added to 200 μL of 0.25 M BLG-NCA in dichloromethane solution to carry out the first ring-opening polymerization reaction. The polymerization process was monitored by Fourier transform infrared spectroscopy. After the BLG-NCA monomer signal disappeared, the polymer product was obtained. The polymer product is a macromolecule with steric hindrance, α-helical structure and terminal amino activity, labeled as G3-PBLG. 50 (m=50,n=0). Among them, the dichloromethane solution of BLG-NCA is obtained by dissolving BLG-NCA monomer in dichloromethane, and its concentration is 0.25M; the molar ratio of BLG-NCA monomer to initiator G3-PAMAM [M]0 / [I]0 is 50:1, where [M]0 is the molar amount of BLG-NCA monomer and [I]0 is the molar amount of initiator.

[0091] 2) Second ring-opening polymerization: Add 200 μL of 0.25 M Val-NCA dichloromethane solution to the polymerization product obtained in 1) to carry out the second ring-opening polymerization reaction for 5 min until the reaction is complete, yielding the block copolymer polypeptide (G3-PBLG). 50 -b-PVal 50 A dichloromethane solution of (m=50, n=50) was designated as the experimental group. The dichloromethane solution of Val-NCA was obtained by dissolving Val-NCA monomer in dichloromethane. The solid content of the block copolymer peptide solution obtained in this example was 60 wt%.

[0092] Meanwhile, a control group (PAMAM group) was set up, including the following:

[0093] 1.25 μL of G3-PAMAM was directly added to 200 μL of 0.25 M Val-NCA dichloromethane solution to carry out ring-opening polymerization for 10 min to obtain the reaction product.

[0094] The results showed that in the control group (PAMAM group), precipitation occurred after 10 minutes of reaction. The terminal amino group of Val-NCA was encapsulated by the precipitation, resulting in terminal deactivation and the inability to continue the ring-opening polymerization reaction.

[0095] The products obtained from the experimental group and the control group were subjected to infrared spectroscopy tests, and the results are shown in Figure 1.

[0096] As shown in Figure 1, the amide I region of the experimental group is mainly located at 1650 cm⁻¹. -1 Near the α-helical conformation, the amide I region of the control group is mainly located at 1635 cm⁻¹. -1 Nearby, the precipitate exhibits a β-sheet conformation. Infrared detection revealed two characteristic peaks (1850 cm⁻¹) of the Val-NCA monomer that persisted for an extended period in the polymerization product. -1 and 1790cm -1 This indicates that after precipitation, the amino activity is encapsulated by the precipitate, leading to deactivation, and the monomer can no longer react.

[0097] The above experimental results indicate that introducing the dendritic helical macromolecule PAMAM-PBLG into the N-carboxylate anhydride of the second amino acid, which readily forms a β-sheet conformation, is effective. 50 This method effectively induces polypeptides that should form a β-sheet conformation to grow in a metastable helical form. This approach avoids the precipitation of the N-carboxylic anhydride of the second amino acid, which is prone to forming a β-sheet conformation, during ring-opening polymerization. It solves the common problem of premature chain termination in the reaction of the N-carboxylic anhydride of the second amino acid with a β-sheet conformation and dendritic polymers, thus facilitating the subsequent synthesis of alternating multiblock polymers.

[0098] Example 2

[0099] Based on Example 1, Example 2 involves selecting appropriate NCA monomers and performing multiple ring-opening polymerization reactions to ultimately obtain a block copolymer polypeptide with alternating α-helical and β-sheet secondary structures. The preparation method is shown in Figure 2 and includes the following steps:

[0100] 1) First ring-opening polymerization reaction: 0.5 μL of G3-PAMAM was added to 400 μL of 0.05 M BLG-NCA in dichloromethane solution to carry out the first ring-opening polymerization reaction. The process was monitored by Fourier transform infrared spectroscopy. After the BLG-NCA monomer signal disappeared, the polymer product, namely G3-PBLG with steric hindrance, α-helical structure and terminal amino activity, was obtained. 50 (m=50,n=0). Among them, the dichloromethane solution of BLG-NCA is obtained by dissolving BLG-NCA monomer in dichloromethane; the molar ratio of BLG-NCA monomer to initiator G3-PAMAM [M]0 / [I]0 is 50:1.

[0101] 2) Second ring-opening polymerization: 400 μL of a 0.05 M solution of BLT-NCA in dichloromethane was added to the polymerization product from step 1) to initiate a second ring-opening polymerization reaction. The process was monitored using Fourier transform infrared spectroscopy. After the BLT-NCA monomer signal disappeared, the block copolymer peptide was successfully prepared and labeled as G3-PBLG.50 -b-PBLT 50 (m=50, n=50). The dichloromethane solution of BLT-NCA is obtained by dissolving BLT-NCA monomer in dichloromethane.

[0102] 3) Third ring-opening polymerization: 400 μL of a 0.25 M solution of BLG-NCA in dichloromethane was added to the polymerization product obtained in step 2) to carry out a third ring-opening polymerization reaction. The block copolymer obtained from the third ring-opening polymerization reaction was labeled as G3-PBLG. 50 -b-(PBLT 50 -PBLG 50 )1(m=50, n=50).

[0103] 4) Fourth ring-opening polymerization: 400 μL of a 0.25 M solution of BLT-NCA in dichloromethane was added to the polymerization product obtained in step 3) to carry out the fourth addition polymerization reaction. The block copolymer peptide obtained from the fourth ring-opening polymerization reaction was labeled as G3-(PBLG) 50 -b-PBLT 50 )2(m=50, n=50).

[0104] 5) Fifth ring-opening polymerization: Repeat step 3) once with the polymerization product obtained in step 4). The block copolymer peptide obtained from the fifth ring-opening polymerization is labeled G3-PBLG. 50 -b-(PBLT 50 -PBLG 50 )2(m=50, n=50).

[0105] 6) Sixth ring-opening polymerization: Repeat step 4) once with the polymerization product obtained in 5). The block copolymer obtained from the sixth ring-opening polymerization is labeled as G3-(PBLG). 50 -b-PBLT 50 )3(m=50, n=50).

[0106] 7) Seventh ring-opening polymerization: Repeat step 3) once with the polymerization product obtained in 6); the block copolymer obtained from the seventh ring-opening polymerization is labeled as G3-PBLG. 50 -b-(PBLT 50 -PBLG 50 )3(m=50, n=50).

[0107] 8) Eighth ring-opening polymerization: Repeat step 4) once with the polymer obtained in 7). The block copolymer obtained from the eighth ring-opening polymerization is labeled as G3-(PBLG). 50 -b-PBLT 50 )4(m=50, n=50).

[0108] Purification: The products G3-PBLG obtained from the 1st, 3rd, 5th and 7th ring-opening polymerization reactions were purified. 50 G3-PBLG 50 -b-(PBLT 50 -PBLG 50 1. G3-PBLG 50 -b-(PBLT 50 -PBLG 50 2. G3-PBLG 50 -b-(PBLT 50 -PBLG 50 3. Add the product to diethyl ether, collect the precipitate, and purify it to obtain a white precipitate. Then, centrifuge the white precipitate and dry it in a vacuum drying oven at 25°C to obtain the purified block copolymer polypeptide.

[0109] The purified block copolymer was dissolved in N,N-dimethylformamide (DMF) solution containing 0.1M lithium bromide, filtered through a 0.22 μm polytetrafluoroethylene membrane, and then detected by gel permeation chromatography (GPC, Thermo Fisher Scientific, 50 Waltham, MA, USA). The results are shown in Figure 3.

[0110] The GPC curves for block copolymers with different segments are shown in Figure 3A, with the GPC-derived light scattering signal as the ordinate (normolizied LS response) and the elution time as the abscissa. The abscissa represents the time required for the block copolymer to pass through the gel permeation chromatography (GPC) column. The ordinate represents the response intensity of the block copolymer.

[0111] As shown in Figure 3A, with the increase of the number of blocks (from G3-PBLG), 50 To G3-PBLG 50 -b-(PBLT 50 -PBLG 50 )3) The GPC curve gradually shifts to the left, indicating that the molecular weight gradually increases. The symmetry and narrow peaks of each curve indicate that the molecular weight distribution is narrow, which means that the synthesized block copolymer peptide has good molecular weight uniformity. The narrow molecular weight distribution is beneficial to the reproducibility of subsequent spinning and block copolymerization.

[0112] Figure 3B is formed by plotting the number of blocks in the block copolymer polypeptide on the x-axis and the weight-average molecular weight (Mw, unit: Da) and polydispersity index (PDI) on the y-axis.

[0113] As shown in Figure 3B, the red curve indicates that the molecular weight increases linearly with the increase of the number of blocks, indicating that the addition of blocks at each step significantly increases the molecular weight of the block copolymer. The blue scatter plots show that the PDI value increases slightly with the increase of the number of blocks but remains at a low level (about 1.0 to 1.2), indicating that the molecular weight distribution of the block copolymer is narrow and uniform.

[0114] As can be seen from Figure 3, this reaction is a living polymerization (the terminal amino group is active), the molecular weight increases linearly, the reaction is controllable, and the molecular weight distribution of the block copolymer is narrow.

[0115] In summary, the stepwise addition of block units enables precise control of molecular weight, which is crucial for designing polymer fibers with specific properties. The narrow molecular weight distribution (low PDI value) indicates good controllability of the synthesis process, ensuring the consistency and predictability of the block copolymer peptide properties.

[0116] Example 3

[0117] This Example 3, based on Example 2, increases the NCA monomer concentration during the reaction to obtain a high-solids-content block copolymer solution without requiring precipitation, purification, reconstitution, concentration, or other similar procedures. Furthermore, the block copolymer solution containing high solids content, high β-sheet content, and high molecular weight can remain stable for a long time without forming a gel.

[0118] 1) First ring-opening polymerization reaction: 2.5 μL of G3-PAMAM was added to 400 μL of 0.25 M BLG-NCA dichloromethane solution to carry out the ring-opening polymerization reaction. The polymerization process was monitored by Fourier transform infrared spectroscopy. After the BLG-NCA monomer signal disappeared, the polymerization product was obtained and labeled as G3-PBLG. 50 The BLG-NCA dichloromethane solution was obtained by dissolving the BLG-NCA monomer in dichloromethane, with a concentration of 0.25 M. The molar ratio of BLG-NCA monomer to initiator G3-PAMAM, [M]0 / [I]0, was 50:1, where [M]0 was the molar amount of BLG-NCA monomer and [I]0 was the molar amount of initiator.

[0119] 2) Second ring-opening polymerization reaction: 400 μL of a 0.25 M dichloromethane solution of BLT-NCA was added to the polymerization product obtained in 1). The polymerization process was monitored using Fourier transform infrared spectroscopy. After the BLT-NCA monomer signal disappeared, the block copolymer polypeptide, namely G3-PBLG with active amino terminals, was obtained. 50 -b-PBLT 50The BLT-NCA dichloromethane solution was obtained by dissolving the BLT-NCA monomer in dichloromethane, and its concentration was 0.25M.

[0120] 3) Repeat the above steps 6 times. After the previous monomer reaction is complete, add the next monomer in sequence. After the BLT-NCA monomer in the 8th stage is consumed, G3-PAMAM-(PBLG) with a solid content of 60wt% is successfully prepared. 50 -b-PBLT 50 4.

[0121] G3-PAMAM-(PBLG) with a solid content of 60 wt% 50 -b-PBLT 50 4. Stability and viscosity tests were performed at room temperature. This included the following steps:

[0122] 250 μL of G3-PAMAM-(PBLG) with a solid content of 60 wt% was collected at days 1, 3, and 15. 50 -b-PBLT 50 4. The strain was measured on a rheometer platform. Strain scanning tests were performed at 25°C with an angular frequency of 5 rad / s within a strain range of 0.1–200%. Oscillatory strain represents the strain level applied to the polymer fiber. The results are shown in Figure 4. The left graph shows the variation of viscosity (Pa.s) with oscillation strain (%), and the right graph shows the variation of modulus (Pa) with oscillation strain. The modulus includes storage modulus (G') and loss modulus (G”), representing elastic and viscous properties, respectively.

[0123] As shown in Figure 4A, the viscosity generally decreases with increasing oscillatory strain, indicating that under larger strain, the viscosity of G3-PAMAM-(PBLG) with a solid content of 60wt% is relatively stable. 50 -b-PBLT 50 4. The fluidity of the solution increases; the viscosity curves on day 1, day 3 and day 15 are relatively similar, indicating that the solution has good stability and the viscosity will not decrease over time.

[0124] As shown in Figure 4B, both G' and G” decrease with increasing oscillation strain, indicating that G3-PAMAM-(PBLG) with a solid content of 60wt% exhibits a decreasing trend. 50 -b-PBLT 50 The elasticity and viscosity of the solution increased under strain; the modulus curves on day 1, day 3 and day 15 were relatively close, indicating that the solution had good stability and would not decrease in elasticity and viscosity over time, and that no further cross-linking structure was formed.

[0125] In summary, G3-PAMAM-(PBLG) within 15 days 50 -b-PBLT 50 The viscosity and modulus of the solution did not change significantly, indicating that the block copolymer peptide solution was stable and that the β-sheet block sequences it contained did not assemble or transform into β-sheets. In contrast, existing technologies report that solutions of natural or other synthetic spider silk or silk readily form hydrogels over time.

[0126] Example 4

[0127] This Example 4, based on Example 2, obtains a block polymer with a lower degree of polymerization by reducing the molar ratio of NCA monomer to initiator during the reaction. This is used to prepare G3-PAMAM-PBLG. 10 -b-PBLT 10 and G3-PAMAM-PBLG 10 -b-PBLT 10 -PBLG 10 Examples will be provided to illustrate this.

[0128] 4.1, G3-PAMAM-PBLG 10 -b-PBLT 10 Preparation

[0129] 1) First ring-opening polymerization reaction: 2.5 μL of G3-PAMAM was added to 80 μL of 0.25 M BLG-NCA in dichloromethane solution to carry out the first ring-opening polymerization reaction. The polymerization process was monitored by Fourier transform infrared spectroscopy. After the BLG-NCA monomer signal disappeared, the polymer product was obtained and labeled as G3-PBLG. 10 (m=10,n=0). Among them, the dichloromethane solution of BLG-NCA is obtained by dissolving BLG-NCA monomer in dichloromethane, and its concentration is 0.25M; the molar ratio of BLG-NCA monomer to initiator G3-PAMAM [M]0 / [I]0 is 10:1, where [M]0 is the molar amount of BLG-NCA monomer and [I]0 is the molar amount of initiator.

[0130] 2) Second ring-opening polymerization: 80 μL of a 0.25 M BLT-NCA solution in dichloromethane was added to the polymerization product obtained in 1) to carry out a second ring-opening polymerization reaction. The polymerization process was monitored using Fourier transform infrared spectroscopy. After the BLT-NCA monomer signal disappeared, the block copolymer polypeptide, namely G3-PBLG with active amino terminals, was obtained. 10 -b-PBLT 10 (m=10, n=10). The BLT-NCA dichloromethane solution was obtained by dissolving the BLT-NCA monomer in dichloromethane, and its concentration was 0.25M.

[0131] 3) Third ring-opening polymerization reaction: 80 μL of 0.25 M BLG-NCA dichloromethane solution was added to the block copolymer obtained in 2) to carry out the third polymerization reaction, and the polymer product was labeled as G3-PBLG. 10 -b-PBLT 10 -PBLG 10 (m = 10, n = 10).

[0132] Purification: The products G3-PBLG obtained from the first and third ring-opening polymerization reactions were purified. 10 G3-PBLG 10 -b-PBLT 10 -PBLG 10 Add the product dropwise to diethyl ether, collect the precipitate, and purify it to obtain a white precipitate. Then, centrifuge the white precipitate and dry it in a vacuum drying oven at 25°C to obtain the purified product.

[0133] The purified product was dissolved in N,N-dimethylformamide (DMF) solution containing 0.1M lithium bromide, filtered through a 0.22 μm polytetrafluoroethylene membrane, and then detected by gel permeation chromatography (GPC, Thermo Fisher Scientific, Waltham, MA, USA). The results are shown in Figure 5.

[0134] As shown in Figure 5, when m and n are 10, the reaction is also controllable. At the same time, this reaction is also a living polymerization (the terminal amino group is active), which can realize the preparation of blocks.

[0135] Example 6: Block copolymer peptides were converted to β-sheets after being treated with DMF.

[0136] This example illustrates that block copolymers contain a large number of β-sheet conformations. In block copolymers, they exist in a metastable helical structure, but can be converted to β-sheet after treatment with DMF for a period of time.

[0137] Take the block copolymer polypeptide solution (G3-PBLG) obtained in Example 2 50 -b-(PBLT 50 -PBLG 50 )4 Solution) was diluted with dichloromethane to a concentration of 1 mg / mL block copolymer solution.

[0138] Take 200 μL of a 1 mg / mL block copolymer dilution solution, then add 50 μL of DMF and mix. After 2 hours, the changes in secondary structure before and after DMF treatment were detected by infrared spectroscopy. The results are shown in Figure 5. The horizontal axis represents the wavenumber (cm²). -1The vertical axis represents absorbance. The graph contains two curves: one is the spectrum before DMF treatment (PAMA-PBLG). 50 -b-PBLT 50 The spectrum has two interpretations: one is the solution spectrum, and the other is the spectrum treated with DMF. In infrared spectroscopy, the characteristic absorption peak of the β-sheet structure typically appears at approximately 1620-1640 cm⁻¹. -1 Within the range of ), the dashed box marks the β-sheet formation.

[0139] As shown in Figure 6, before the addition of DMF, G3-PAMAM-(PBLG) 50 -b-PBLT 50 The β-sheet conformation in (4) is a metastable helix, which is thermodynamically unstable. The addition of DMF significantly increased the absorbance of the β-sheet region, indicating an increase in the number of β-sheet structures. In summary, the addition of DMF can induce the block copolymer to transform into a thermodynamically stable β-sheet. Furthermore, methanol was also found to induce the transformation of the metastable helix in the block copolymer into a thermodynamically stable β-sheet.

[0140] Example 7

[0141] The difference between this embodiment and Example 3 is that the amino acid in the second amino acid N-carboxylic acid anhydride is replaced with valine (Val-NCA), while the rest is the same as in Example 3, resulting in the block copolymer G3-PAMAM-(PBLG). 50 -b-PVal 50 4.

[0142] Example 8

[0143] The difference between this embodiment and Example 3 is that the amino acid in the second amino acid N-carboxylic acid anhydride is changed to phenylalanine (Phe-NCA), while the rest is the same as in Example 3, resulting in the block copolymer G3-PAMAM-(PBLG). 50 -b-PPhe 50 4.

[0144] Example 9

[0145] The difference between this embodiment and Example 3 is that the amino acid in the second amino acid N-carboxylic acid anhydride is replaced with serine (Ser-NCA), while the rest is the same as in Example 3, resulting in the block copolymer G3-PAMAM-(PBLG). 50 -b-PSer 50 4.

[0146] Example 10

[0147] The difference between this embodiment and Embodiment 3 is that G1-PAMAM is used as the initiator, while the rest are the same as in Embodiment 3.

[0148] Example 11

[0149] The difference between this embodiment and Embodiment 3 is that G6-PAMAM is used as the initiator, while the rest are the same as in Embodiment 3.

[0150] Application Example 1

[0151] In this embodiment, the G3-PAMAM-(PBLG) with a solid content of 60 wt% obtained in Example 3 is used. 50 -b-PBLT 50 4. Solution preparation to form polymer fibers, also known as spider silk fibers. This includes the following steps:

[0152] 1) Amorphous fibers are prepared by self-assembly using a rapid injection molding process.

[0153] Using a methanol / DMF mixture of 3:1 as the coagulation bath, G3-PAMAM-(PBLG) with a solid content of 60wt% was rapidly sheared (20 ml / min) to remove the solids. 50 -b-PBLT 50 )4 The solution is squeezed into the coagulation bath to assemble it into amorphous fibers.

[0154] 2) Strain-programmed crystallization of amorphous fibers

[0155] By fixing both ends of the amorphous fiber in step 1), stretching it at a uniform speed of 2 mm / s by different multiples and drying it under constant strain conditions at both ends, high-strength and high-toughness polymer fibers containing a large number of β-folded crystals with different degrees of strain programming were obtained. The fiber samples (polymer fibers) obtained by stretching 0 times (i.e., no stretching), 1 time, and 2 times were designated as group 0X, group 1X, and group 2X, respectively.

[0156] Morphological observation, cross-sectional and longitudinal microstructure analysis, crystallographic observation, characterization of the secondary structure of the fibers, and mechanical property testing were performed on fiber samples with different draw ratios.

[0157] 1-1) Macroscopic morphological observation

[0158] As shown in Figure 7, the unstretched amorphous fiber samples (Group 0X) exhibit a milky white, slender fiber morphology. After stretching, taking Group 2X as an example, the fiber samples became longer and thinner, and showed a reflective crystalline luster. This indicates that before stretching, the coagulation bath mainly induced the polymer fibers to solidify and assemble, while stretching induced the fiber orientation and crystallization.

[0159] 1-2) Microstructure of cross-section and longitudinal section

[0160] Fiber samples with different draw ratios were adhered to the sample stage using double-sided conductive tape. A 3 nm thick layer of platinum was sputtered onto the sample surface using a Leica sputtering instrument to achieve conductivity. The microstructure of the cross-section and longitudinal section of the fiber samples was then observed using a field emission scanning electron microscope. The test results are shown in Figures A and B in Figure 8.

[0161] As shown in Figure 8A, the cross-section of the unstretched fiber sample (Group 0X) is a disordered packing, forming an irregular interface, and the overall structure lacks directional arrangement characteristics. The cross-section of the fiber sample with 1x stretch (Group 1X) shows significant deformation and reorganization, transforming the original disordered packing into a clear directional arrangement. The cross-section of the fiber sample with 2x stretch (Group 2X) shows further oriented arrangement along the stretching direction, and the packing is more tightly integrated.

[0162] Figure 8B shows the longitudinal cross-sectional morphology of the fiber sample (2X group) after double stretching and its high-resolution magnified local image.

[0163] As shown in Figure 8B, a single fiber appears as a uniform cylinder; magnified observation reveals that it is composed of hundreds of microscopic nanofibers with diameters ranging from 50 to 200 nanometers, arranged parallel to each other along the axial direction (i.e., a multi-layered structure) forming a bundle-like structure. In summary, multi-layered artificial spider silk can be prepared using wet spinning technology and post-drawing processes, and drawing can induce a regular orientation of amorphous fibers.

[0164] 1-3) Crystallographic observation

[0165] Fiber samples with different stretch ratios were fixed at both ends and attached to glass slides for observation under polarizing microscopy on a vertical Zeiss Axiovert 200M inverted microscope. Polarizing microscopy images of the fibers were collected at a polarization angle of 90° relative to the analyzer. The test results are shown in Figure 9.

[0166] As shown in Figure 9, the fiber samples in group 0X exhibit no significant optical path refraction under crossed polarized light, indicating that most of the polymer fibers are amorphous and lack crystal formation. The fiber samples in group 1X display bright, continuous orange and green interference bands along the stretching direction under crossed polarized light, with no extinction dark areas between the bands, proving that the crystals are highly oriented within the fiber cross-section, forming continuously distributed liquid crystal domains without significant phase separation. The fiber samples in group 2X display only a single blue interference color along the stretching direction under crossed polarized light, and the band width is significantly increased.

[0167] Furthermore, multiple parallel fibers were tightly wound onto a specially designed sample holder, ensuring that the fibers were substantially parallel to the rotation axis of the goniometer. The crystal unit structure and orientation of the fibers were measured by wide-angle X-ray scattering (WAXS) using a DUO micro-source single-crystal X-ray diffractometer with collimating optics and a two-dimensional X-ray detector (SCXRD, Bruker GmbH, Germany). The fiber sample was placed vertically and exposed for 10 seconds under ambient conditions using a 13.50 kEV beam (wavelength 0.9184 Å). The scattering images were captured by a Decris Pilatus 2M detector, as shown in Figure 10.

[0168] As shown in Figure 10, the fiber samples in group 0X exhibit diffuse scattering rings, indicating an amorphous structure. The fiber samples in groups 1X and 2X, on the other hand... (β chain spacing) and The presence of distinct diffraction rings (interlayer spacing) confirms the presence of parallel, large β-folded sheet crystals in the fiber sample.

[0169] In summary, the stretching process can induce the formation of β-folded nanocrystals, and the crystal dispersion becomes more uniform as the stretching ratio increases.

[0170] 1-4) The secondary structure of the fiber was characterized using infrared spectroscopy.

[0171] The secondary structure of the fiber samples was characterized using Fourier transform infrared spectroscopy (equipped with an attenuated total reflectance infrared spectroscopy device (ATR)). Fiber samples prepared at different draw ratios (0X group, 1X group, 2X group) were placed on the ATR for infrared spectral data acquisition (scanning range 4000-400 cm⁻¹). -1 The scanning resolution is 4cm. -1 The scans were performed 16 times. Then, the second derivative method was used to perform peak deconvolution integrals on the amide I region of different fiber samples, and a Gaussian model was established to estimate the content of various secondary structures in fiber samples with different draw ratios (see J. Am. Chem. Soc 2024, 146, 46, 31849–31859 ​​for fitting references). The infrared measurement results and fitting analysis results are shown in Figure 11. The Amide I region (approximately 1600-1700 cm⁻¹) is mainly used to analyze the secondary structure of proteins or peptides.

[0172] As shown in Figure 11A, the absorption peak positions and intensities in the amide I region of fiber samples with different draw ratios (0X group, 1X group, and 2X group) differ significantly. With increasing draw ratio, the spectra at β-fold (approximately 1620-1640 cm⁻¹) show significant differences. -1 ), α-helix (approximately 1650-1660 cm) -1 ), random curls (approximately 1640-1650cm)-1 ) and β-turn (approximately 1660-1700cm) -1 The absorbance at characteristic peaks such as α-helix changed. Compared with group 0X, the absorbance of fiber samples in groups 1X and 2X increased in the β-fold region, indicating that the stretching treatment promoted the formation of the β-fold structure. At the same time, the absorbance of the α-helix decreased slightly, indicating that the metastable α-helix structure transformed into a β-fold structure.

[0173] As shown in Figure 11B, the curves of different colors represent the characteristic peaks of secondary structures such as β-sheets, α-helices, random coils, and β-turns. With increasing draw ratio, β-sheets (βsheet, 1620-1640 cm⁻¹) exhibit characteristic peaks. -1 The peak intensity corresponding to α-helix (1650-1660 cm⁻¹) is significantly enhanced; while the peak intensity of α-helix (1650-1660 cm⁻¹) is significantly enhanced. -1 The characteristic peaks of ) are relatively weakened; random coil (Coil, 1640-1650 cm⁻¹) -1 The peak of the ) weakened. This further confirms the effect of the stretching treatment on the transformation of the secondary structure.

[0174] As shown in Figure 11C, the content of each secondary structure was quantified by peak fitting, verifying the increasing trend of β-sheet content.

[0175] With increasing draw ratio, the relative content of β-sheets increased significantly, from approximately 0% in the 0X group to 40% in the 1X group and 60% in the 2X group. Simultaneously, the relative content of α-helices decreased from approximately 80% in the 0X group to approximately 20% in the 2X group, and the relative content of β-turns also decreased. Drawing treatment significantly promoted the formation of β-sheet structures in polymer fibers while reducing the content of α-helices and β-turns. This indicates that drawing treatment can effectively regulate the secondary structure of fiber samples, thereby altering their physical and chemical properties.

[0176] In summary, the drawing process can induce the transformation of β-sheet blocks in polymer fibers, which originally existed in a metastable α-helix state, into thermodynamically more stable β-sheet crystals, while reducing the content of α-helices and β-turns. Furthermore, with the increase of the drawing ratio, the molecular chains become more tightly packed, and the β-sheet content increases significantly.

[0177] 1-5) Mechanical property testing

[0178] Mechanical properties of fiber samples prepared with different draw ratios (0X, 1X, 2X, and 3X groups) were tested using a tensile strength tester (CellScale, UniVert, Canada). After observing and recording the diameter of each fiber under an optical microscope, tensile tests were performed on 4mm long fiber samples at a stretching rate of 50mm / min until the sample broke. Then, stress-strain curves were plotted based on the cross-sectional area and length to obtain mechanical properties such as tensile strength and strain. At least five samples were tested for each fiber group. The results are shown in Figure 12. In Figure 12, the horizontal axis represents strain (%), and the vertical axis represents tensile strength. The red dashed box indicates that under this stress, chain slippage occurs between the polymer fiber molecular chains, moving away from initial chain entanglement. The maximum tensile strength of the fiber sample can be obtained from the stress-strain curve, i.e., the value corresponding to the highest point of the curve, reflecting the maximum tensile strength that the polymer fiber can withstand before breakage. In B, the horizontal axis represents the draw ratio, and the vertical axis represents the tensile strength (stress) and strain (strain).

[0179] As shown in the stress-strain curves of Figure 12A, the tensile strength of the fiber samples increases with the increase of the draw ratio. The tensile strength of the 0X group is about 50 MPa, the tensile strength of the 1X group is about 100 MPa, the tensile strength of the 2X group is about 175 MPa, and the tensile strength of the 3X group can reach about 200 MPa.

[0180] As shown in Figure 12B, the tensile strength of the polymer fiber increases significantly with the increase of the draw ratio, from approximately 50 MPa in the 0X group to approximately 225 MPa in the 3X group. Meanwhile, the strain decreases significantly with the increase of the draw ratio, from approximately 140% in the 0X group to approximately 30% in the 3X group.

[0181] In summary, the stretching treatment significantly improved the tensile strength of polymer fibers, indicating that structural rearrangement or reinforcement occurred during the stretching process. As the stretching ratio increased, the ductility (manifested as strain) of polymer fibers decreased significantly, indicating that the polymer fibers became brittle.

[0182] Application Example 2

[0183] This application example investigated the effects of different amino acid N-carboxyl anhydrides and different polypeptide chain ends on mechanical properties.

[0184] 2.1 Effects of different amino acids on mechanical properties

[0185] G3-PAMAM-(PBLG) with a solid content of 60 wt% obtained in Examples 7-9 were used respectively. 50 -b-PVal50 4. G3-PAMAM-(PBLG) 50 -b-PPhe 50 4. G3-PAMAM-(PBLG) 50 -b-PSer 50 Solution 4 was prepared using the same method as in Application Example 1 to form corresponding fiber samples of 2X, and mechanical properties were tested. The mechanical properties are shown in Table 1 below. Simultaneously, G3-PAMAM-(PBLG) with a solid content of 60 wt% obtained in Example 3 was also tested. 50 -b-PBLT 50 The corresponding fiber samples of the 2X group formed by )4 (that is, the 2X fiber samples obtained by application example 1) are listed in Table 1.

[0186] Table 1

[0187] As shown in Table 1, the tensile strength of the polymer fibers formed by the block copolymers of the present invention is above 70 MPa.

[0188] 2.2 Effects of different ends on mechanical properties

[0189] The influence of the terminal secondary structure of the polypeptide chain on the mechanical properties of polymer fibers was verified. The results showed that the polymer fibers exhibited optimal mechanical properties when the first block was an α-helical tendency structure (PBLG) and the terminal block was a β-sheet tendency structure (PBLT).

[0190] Spinning solution G3-(PBLG) for forming polymer fibers was prepared according to the method in Example 2. 50 -b-PBLT 50 )4, and further ring-opening polymerization was carried out on it to obtain G3-(PBLG) 50 -b-PBLT 50 )4-b-PBLG 50 :

[0191] G3-(PBLG 50 -b-PBLT 50 )4 (The first segment of the NCA connected to PAMAM contains an α-helix, and the last segment contains a β-fold, denoted as α-β).

[0192] G3-(PBLG 50 -b-PBLT 50 )4-b-PBLG 50 (The NCA connected to PAMAM contains an α-helix in both its initial and final segments, denoted as α-α). This is also known as G3-PBLG. 50 -b-(PBLT 50 -PBLG50 Add 400 μL of 0.25 M BLG-NCA dichloromethane solution to G3 to carry out ring-opening polymerization to obtain G3-(PBLG) 50 -b-PBLT 50 )4-b-PBLG 50 .

[0193] G3-(PBLT 50 -b-PBLG 50 4 (The NCA linked to PAMAM contains a β-sheet at the beginning and an α-helix at the end, denoted as β-α). Its preparation method is as follows: 0.5 μL of G3-PAMAM is added to 400 μL of a 0.25 M BLT-NCA solution in dichloromethane for the first ring-opening polymerization reaction to obtain the polymer product. Then, 400 μL of a 0.25 M BLG-NCA solution in dichloromethane is added for the second ring-opening polymerization reaction; this process is repeated three times to obtain G3-PAMAM-(PBLT-NCA). 50 -b-PBLG 50 4.

[0194] G3-(PBLT 50 -b-PBLG 50 )3-b-PBLT 50 (The NCA connected to PAMAM contains β-sheets in both the first and last segments, denoted as β-β). The preparation method is as follows: 0.5 μL of G3-PAMAM is added to 400 μL of a 0.25 M BLT-NCA solution in dichloromethane for the first ring-opening polymerization reaction to obtain the polymer product. Then, 400 μL of a 0.25 M BLG-NCA solution in dichloromethane is added for the second ring-opening polymerization reaction; this process is repeated twice to obtain G3-(PBLT-NCA). 50 -b-PBLG 50 4. Then, add 400 μL of a 0.05 M solution of BLT-NCA in dichloromethane to carry out a ring-opening polymerization reaction to obtain G3-(PBLT) 50 -b-PBLG 50 )3-b-PBLT 50 .

[0195] Polymer fibers with a draw ratio of 2X were obtained by using the process described in Application Example 1. Mechanical properties were tested according to the method described in Application Example 1, and the results are shown in Figure 13.

[0196] As shown in Figure 13A, the tensile strength ranking is α-β (175MPa) > β-β (125MPa) > β-α (99MPa) > α-α (96MPa).

[0197] As shown in Figure 13B, the strain order is α-β (40%) > β-β (35%) > β-α (22%) > α-α (11%). These results demonstrate that the presence of a β-folded block at the end significantly improves strength (α-β is 82% stronger than α-α). The presence of an α-helical block at the beginning effectively enhances ductility. The α-β combination maximizes the fracture energy, achieving a synergistic effect of strength and toughness. In summary, the sequence regulation of the dual-end secondary structure can significantly optimize the mechanical properties of polymer fibers, with the α-helix at the beginning / β-fold at the end (α-β) sequence design being the optimal solution.

[0198] Comparative Example 1

[0199] In this comparative example, a block copolymer polypeptide was prepared using the first amino acid N-carboxylic acid anhydride and G3-PAMAM as raw materials. It contained only structural units that were easy to form α-helices. Then, it was spun using the same process as in Application Example 1.

[0200] 1) Preparation of G3-PAMAM-PBLG by ring-opening polymerization 400 solution

[0201] 1.25 μL of G3-PAMAM was added to 1.6 mL of a 0.25 M solution of BLG-NCA in dichloromethane to initiate a ring-opening polymerization reaction. The process was monitored using Fourier transform infrared spectroscopy. After the BLG-NCA monomer signal disappeared, the polymer G3-PAMAM-PBLG was synthesized. 400 The polymerized product exhibits steric hindrance and an α-helical structure. The BLG-NCA dichloromethane solution was obtained by dissolving the BLG-NCA monomer in dichloromethane; the molar ratio of BLG-NCA monomer to initiator G3-PAMAM [M]0 / [I]0 was 400:1. The solid content of the polymerized product in this comparative example was 60 wt%.

[0202] 2) G3-PAMAM-PBLG 400 Wet spinning process

[0203] Using a methanol / DMF mixture of 3:1 as the coagulation bath, G3-PAMAM-PBLG with a solid content of 60 wt% was coagulated by rapid shearing (20 ml / min). 400 The solution is squeezed into the coagulation bath to assemble it into amorphous fibers.

[0204] G3-PAMAM-PBLG 400 Figure 14 shows a real shot of the solution dissolved in the coagulation bath.

[0205] As shown in Figure 14, G3-PAMAM-PBLG 400The fibers were assembled into loose, flocculent white fibers in the coagulation bath, failing to form stable fibers, and thus unable to undergo subsequent drawing processes or form fibers with high mechanical properties.

[0206] The above results indicate that β-sheet blocks are essential structural units for wet spinning to prepare high-performance fibers, while pure α-helical systems cannot achieve stable molding due to the lack of effective crosslinking sites.

[0207] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A copolymer polypeptide, characterized in that, The invention comprises a dendritic polymer and a polypeptide chain connected to the dendritic polymer, the polypeptide chain being composed of structural units shown in Formula I and Formula II, the dendritic polymer being connected to the structural unit shown in Formula I via amide bonds, and the structural unit shown in Formula I being connected to the structural unit shown in Formula II via amide bonds. In Formula I, R1 is selected from the side groups of the following amino acids: glutamic acid, lysine, arginine, glutamine, and methionine or their derivatives; In Formula II, R2 is selected from the side groups of the following amino acids: alanine, serine, tyrosine, phenylalanine, valine, threonine, tryptophan, and isoleucine or their derivatives.

2. The block copolymer polypeptide according to claim 1, characterized in that, The general formula of the block copolymer polypeptide is as follows: Where m is independently 10 to 1000, and n is independently 10 to 1000; R is a dendritic polymer.

3. The block copolymer polypeptide according to claim 2, characterized in that, The dendritic polymer is selected from one or more of polyamide-amine, polypropylene imine, and branched polyethyleneimine; And / or, the weight-average molecular weight of the block copolymer is 1 to 10,000 kDa.

4. The method for preparing the block copolymer polypeptide according to any one of claims 1-3, characterized in that, Includes the following steps: 1) The dendritic polymer and the first amino acid N-carboxylic acid anhydride undergo a first ring-opening polymerization reaction in the first solvent to obtain the polymer product; 2) The polymerization product and the second amino acid N-carboxylic anhydride undergo a second ring-opening polymerization reaction in a second solvent to obtain the block copolymer polypeptide; The amino acid in the first amino acid N-carboxylic acid anhydride is selected from glutamic acid, lysine, arginine, glutamine, and methionine or their derivatives; The amino acid in the second amino acid N-carboxylic acid anhydride is selected from alanine, serine, tyrosine, phenylalanine, valine, threonine, tryptophan, and isoleucine or their derivatives.

5. The preparation method according to claim 4, characterized in that, Includes at least one of the following technical features: A1) The first solvent is selected from one or both of dichloromethane and chloroform; A2) The second solvent is selected from one or both of dichloromethane and chloroform; A3) The molar mass ratio of the dendritic polymer and the first amino acid N-carboxylic acid anhydride is 1:(15-400); A4) The molar mass ratio of the dendritic polymer and the second amino acid N-carboxylic acid anhydride is 1:(15-400); A5) The temperature of the first ring-opening polymerization reaction is 10℃~65℃; A6) The temperature of the second ring-opening polymerization reaction is 10℃~65℃.

6. A polymer fiber, characterized in that, The raw materials used in the preparation include the block copolymer polypeptide as described in any one of claims 1-3.

7. The method for preparing polymer fibers as described in claim 6, characterized in that, The block copolymer polypeptide solution is used to obtain the polymer fiber through a spinning process.

8. The preparation method according to claim 7, characterized in that, Includes the following steps: The block copolymer polypeptide solution is mixed into a coagulation bath, and then drawn to obtain the polymer fiber.

9. The preparation method according to claim 8, characterized in that, The coagulation bath is selected from one or more of methanol, N,N-dimethylformamide, ethanol, tetrahydrofuran, ethyl acetate, dimethyl sulfoxide, dichloromethane, and hexafluoroisopropanol; and / or, the solid content of the block copolymer solution is 15-70 wt%. And / or, the stretching factor is 0.5 to 5.0 times.

10. Use of the block copolymer polypeptide of any one of claims 1-3 or the polymer fiber of claim 6 in fiber articles, biomedical materials or drug delivery.

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